In the vast majority of biologically relevant cases of receptor-ligand complex formation, the binding site of the receptor is a small part of its surface, and moreover, formation of a biologically ...active complex often requires a specific orientation of the ligand relative to the binding site. Before the formation of the initial form of the complex, only long-range, electrostatic and hydrodynamic interactions can act between the ligand approaching the binding site and the receptor. In this context, the question arises whether as a result of these interactions, there is a pre-orientation of the ligand towards the binding site, which to some extent would accelerate the formation of the complex. The role of electrostatic interactions in the orientation of the ligand relative to the binding site of the receptor is well documented. The analogous role of hydrodynamic interactions, although assessed as very significant by Brune and Kim (PNAS 91, 2930–2934, (1994)), is still debatable. In this article, I present the current state of knowledge on this subject and consider the possibilities of demonstrating the orienting effect of hydrodynamic interactions in the processes of receptor–ligand association, in an experimental way supported by computer simulations.
In Part 2 we discuss application of several different types of UV–Vis spectroscopy, such as normal, difference, and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and ...circular dichroism spectroscopy, and Raman spectroscopy, of the side-chain of tyrosine residues in different molecular environments. We review the ways these spectroscopies can be used to probe complex protein structures.
Using stopped-flow fluorometry, we determined rate constants for the formation of diffusional encounter complexes of tri-N-acetylglucosamine (NAG3) with hen egg-white lysozyme (k a WT) and its double ...mutant Asp48Asn/Lys116Gln (k a MT). We defined binding anisotropy, κ ≡ (k a WT – k a MT)/(k a WT + k a MT), and determined its ionic strength dependence. Our goal was to check if this ionic strength dependence provides information about the orienting hydrodynamic effects in the ligand-binding process. We also computed ionic strength dependence of the binding anisotropy from Brownian dynamics simulations using simple models of the lysozyme–NAG3 system. The results of our experiments indicate that in the case of lysozyme and NAG3 such hydrodynamic orienting effects are rather negligible. On the other hand, the results of our Brownian dynamics simulations prove that there exist molecular systems for which such orienting effects are substantial. However, the ionic strength dependence of the rate constants for the wild-type and modified systems do not exhibit any qualitative features that would allow us to conclude the presence of hydrodynamic orienting effects from stopped-flow experiments alone. Nevertheless, the results of our simulations suggest the presence of hydrodynamic orienting effects in the receptor–ligand association when the anisotropy of binding depends on the solvent viscosity.
In this work, we investigated the kinetics of binding of hen egg-white lysozyme with tri-N-acetylglucosamine in aqueous solutions, at two values of pH, 3.2 and 11, as a function of ionic strength, by ...a stopped-flow method with tryptophyl fluorescence observation of the transients. We analyzed registered reaction progress curves by employing numerical integration of appropriate chemical master equations. We discriminated between several binding models and established that the process observed in experiments follows a two-step mechanism, composed of four elementary stages: diffusional formation of an encounter complex, dissociation of the encounter complex, conformational transition of the encounter complex to the final complex, and the reverse transformation, i.e., from the final complex to the encounter complex. We evaluated rate constants of these elementary stages and determined their dependencies on solution ionic strength. Regardless of solution pH, rate constants of both forward and reverse conformational transitions increase with an increasing ionic strength. This suggests that ionic screening of intramolecular electrostatic interactions may act to lower the activation barrier for conformational transition in proteins.
The 8-azapurines, and their 7-deaza and 9-deaza congeners, represent a unique class of isosteric (isomorphic) analogues of the natural purines, frequently capable of substituting for the latter in ...many biochemical processes. Particularly interesting is their propensity to exhibit pH-dependent room-temperature fluorescence in aqueous medium, and in non-polar media. We herein review the physico-chemical properties of this class of compounds, with particular emphasis on the fluorescence emission properties of their neutral and/or ionic species, which has led to their widespread use as fluorescent probes in enzymology, including enzymes involved in purine metabolism, agonists/antagonists of adenosine receptors, mechanisms of catalytic RNAs, RNA editing, etc. They are also exceptionally useful fluorescent probes for analytical and clinical applications in crude cell homogenates.
The reaction kinetics between like-charged compounds in water is extremely slow due to Coulomb repulsions. Here, we demonstrate that by screening these interactions and, in consequence, increasing ...the local concentration of reactants, we boost the reactions by many orders of magnitude. The reaction between negatively charged Coenzyme A molecules accelerates ~5 million-fold using cationic micelles. That is ~10
faster kinetics than in 0.5 M NaCl, although the salt is ~10
more concentrated. Rate enhancements are not limited to micelles, as evidenced by significant catalytic effects (10
-10
-fold) of other highly charged species such as oligomers and polymers. We generalize the observed phenomenon by analogously speeding up a non-covalent complex formation-DNA hybridization. A theoretical analysis shows that the acceleration is correlated to the catalysts' surface charge density in both experimental systems and enables predicting and controlling reaction rates of like-charged compounds with counter-charged species.
Proper treatment of hydrodynamic interactions is of importance in evaluation of rigid-body mobility tensors of biomolecules in Stokes flow and in simulations of their folding and solution ...conformation, as well as in simulations of the translational and rotational dynamics of either flexible or rigid molecules in biological systems at low Reynolds numbers. With macromolecules conveniently modeled in calculations or in dynamic simulations as ensembles of spherical frictional elements, various approximations to hydrodynamic interactions, such as the two-body, far-field Rotne–Prager approach, are commonly used, either without concern or as a compromise between the accuracy and the numerical complexity. Strikingly, even though the analytical Rotne–Prager approach fails to describe (both in the qualitative and quantitative sense) mobilities in the simplest system consisting of two spheres, when the distance between their surfaces is of the order of their size, it is commonly applied to model hydrodynamic effects in macromolecular systems. Here, we closely investigate hydrodynamic effects in two and three-body systems, consisting of bead–shell molecular models, using either the analytical Rotne–Prager approach, or an accurate numerical scheme that correctly accounts for the many-body character of hydrodynamic interactions and their short-range behavior. We analyze mobilities, and translational and rotational velocities of bodies resulting from direct forces acting on them. We show, that with the sufficient number of frictional elements in hydrodynamic models of interacting bodies, the far-field approximation is able to provide a description of hydrodynamic effects that is in a reasonable qualitative as well as quantitative agreement with the description resulting from the application of the virtually exact numerical scheme, even for small separations between bodies.
Spectroscopic properties of tyrosine residues may be employed in structural studies of proteins. Here we discuss several different types of UV–Vis spectroscopy, like normal, difference and ...second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, and corresponding optical properties of the tyrosine chromophore, phenol, which are used to study protein structure.
Brownian dynamics (BD) simulations and the first-passage-time approach are applied to investigate diffusion-controlled association in a biologically relevant model system consisting of a fixed ...receptor with an elongated cavity and a capsule-like ligand that fits this cavity precisely. Before the binding at the receptor cavity, the ligand undergoes translational and rotational diffusion, either free or under the influence of electrostatic interactions with the receptor. The spatial dependence of the translational and rotational mobilities of the ligand resulting from its hydrodynamic interactions (HIs) with the receptor is accounted for in BD simulations, and an accurate numerical approach is applied for the evaluation of the spatially dependent mobility tensor of the ligand. Different magnitudes of electrostatic interactions (either attraction or repulsion) between the ligand and receptor are considered. The effective range of receptor–ligand electrostatic interactions is varied to account for their screening under different conditions of ionic strength. The effects of HIs on the kinetics of the diffusion-controlled association, evaluated for different electrostatic properties of binding partners, are thoroughly analyzed and discussed.
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